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Glaucoma is characterized by axonal degeneration of retinal ganglion cells (RGCs) and apoptotic death of their cell bodies. Lowering intraocular pressure is currently the only way to treat glaucoma, but it is often insufficient to inhibit the progression of the disease. Glaucoma is a multifactorial disease, and the involvement of oxidative stress has recently received much attention. In the present study, we investigated the cytoprotective effect of astaxanthin (AST) on RGC degeneration using a normal-tension glaucoma (NTG) mouse model, which lacks theglutamate/aspartate transporter(Glast) and demonstrates spontaneous RGC and optic nerve degeneration without elevated intraocular pressure. Three-week-old Glast(+/-)mice were given intraperitoneal injections of AST at 10, 30, or 60 mg/kg/day or vehicle alone, and littermate control mice were given vehicle alone for 14 days, respectively. Five weeks after birth, the number of RGCs was counted in paraffin sections of retinal tissues stained with hematoxylin and eosin. We also used a retrograde labeling technique to quantify the number of RGCs. Additionally, the phosphorylated (p) I kappa B/total I kappa B ratio and the 4-hydroxynonenal (HNE) were measured in retinal tissues. The number of RGCs in Glast(+/-)mice was significantly decreased compared with that in control mice. RGC loss was suppressed by the administration of AST at 60 mg/kg/day, compared with vehicle alone. Following AST administration, the concentration of 4-HNE in the retina was also suppressed, but the pI kappa B/I kappa B ratio did not change. Our study revealed that the antioxidative stress effects of AST inhibit RGC degeneration in the retina and may be useful in the treatment of NTG.

Glaucoma is characterized by axonal degeneration of retinal ganglion cells (RGCs) and apoptotic death of their cell bodies. Lowering intraocular pressure is currently the only way to treat glaucoma, but it is often insufficient to inhibit the progression of the disease. Glaucoma is a multifactorial disease, and the involvement of oxidative stress has recently received much attention. In the present study, we investigated the cytoprotective effect of astaxanthin (AST) on RGC degeneration using a normal-tension glaucoma (NTG) mouse model, which lacks theglutamate/aspartate transporter(Glast) and demonstrates spontaneous RGC and optic nerve degeneration without elevated intraocular pressure. Three-week-old Glast(+/-)mice were given intraperitoneal injections of AST at 10, 30, or 60 mg/kg/day or vehicle alone, and littermate control mice were given vehicle alone for 14 days, respectively. Five weeks after birth, the number of RGCs was counted in paraffin sections of retinal tissues stained with hematoxylin and eosin. We also used a retrograde labeling technique to quantify the number of RGCs. Additionally, the phosphorylated (p) I kappa B/total I kappa B ratio and the 4-hydroxynonenal (HNE) were measured in retinal tissues. The number of RGCs in Glast(+/-)mice was significantly decreased compared with that in control mice. RGC loss was suppressed by the administration of AST at 60 mg/kg/day, compared with vehicle alone. Following AST administration, the concentration of 4-HNE in the retina was also suppressed, but the pI kappa B/I kappa B ratio did not change. Our study revealed that the antioxidative stress effects of AST inhibit RGC degeneration in the retina and may be useful in the treatment of NTG.

Purpose: The aims of this study were to analyze optical coherence tomography (OCT) imaging of large macular holes (MHs) treated with inverted internal limiting membrane (ILM) flap technique and to perform a histological examination of an ILM-like membrane tissue obtained during vitrectomy. Patients and methods: This is a retrospective observational case study. Nine patients, comprising of five males and four females, showing large and myopic MHs, underwent pars plana vitrectomy (PPV) with inverted ILM flap technique assisted by brilliant blue G (BBG) staining. Ophthalmological findings including visual acuity and OCT were investigated based on medical records. Formalin-fixed paraffin-embedded tissue section of an ILM-like membrane was submitted for immunohistochemistry with glial fibrillary acidic protein (GFAP). Results: ILM was clearly stained with BBG in eight patients, whereas the ILM in one case revealed no staining with BBG during PPV. Visual acuities improved to >0.2 LogMAR in six patients. The complete closure of MH following PPV with inverted ILM technique was eventually achieved in all patients determined by OCT imaging (100%). Only one patient showed recovery of ellipsoid zone and interdigitation zone following the surgery. Elongation of outer nuclear layer was noted in three eyes. The ILM-like membrane not stained with BBG histologically revealed an amorphous structure admixed with GFAP-positive mononuclear cell infiltration. Conclusion: PPV with inverted ILM flap technique achieved 100% closure rates with favorable configuration at an initial surgery in large MHs. Our histopathological data also suggest that even BBG staining-negative membrane may be a useful material for autologous transplantation to the hole.

Purpose: The aims of this study were to analyze optical coherence tomography (OCT) imaging of large macular holes (MHs) treated with inverted internal limiting membrane (ILM) flap technique and to perform a histological examination of an ILM-like membrane tissue obtained during vitrectomy. Patients and methods: This is a retrospective observational case study. Nine patients, comprising of five males and four females, showing large and myopic MHs, underwent pars plana vitrectomy (PPV) with inverted ILM flap technique assisted by brilliant blue G (BBG) staining. Ophthalmological findings including visual acuity and OCT were investigated based on medical records. Formalin-fixed paraffin-embedded tissue section of an ILM-like membrane was submitted for immunohistochemistry with glial fibrillary acidic protein (GFAP). Results: ILM was clearly stained with BBG in eight patients, whereas the ILM in one case revealed no staining with BBG during PPV. Visual acuities improved to >0.2 LogMAR in six patients. The complete closure of MH following PPV with inverted ILM technique was eventually achieved in all patients determined by OCT imaging (100%). Only one patient showed recovery of ellipsoid zone and interdigitation zone following the surgery. Elongation of outer nuclear layer was noted in three eyes. The ILM-like membrane not stained with BBG histologically revealed an amorphous structure admixed with GFAP-positive mononuclear cell infiltration. Conclusion: PPV with inverted ILM flap technique achieved 100% closure rates with favorable configuration at an initial surgery in large MHs. Our histopathological data also suggest that even BBG staining-negative membrane may be a useful material for autologous transplantation to the hole.

Purpose: The aims of this study were to analyze optical coherence tomography (OCT) imaging of large macular holes (MHs) treated with inverted internal limiting membrane (ILM) flap technique and to perform a histological examination of an ILM-like membrane tissue obtained during vitrectomy. Patients and methods: This is a retrospective observational case study. Nine patients, comprising of five males and four females, showing large and myopic MHs, underwent pars plana vitrectomy (PPV) with inverted ILM flap technique assisted by brilliant blue G (BBG) staining. Ophthalmological findings including visual acuity and OCT were investigated based on medical records. Formalin-fixed paraffin-embedded tissue section of an ILM-like membrane was submitted for immunohistochemistry with glial fibrillary acidic protein (GFAP). Results: ILM was clearly stained with BBG in eight patients, whereas the ILM in one case revealed no staining with BBG during PPV. Visual acuities improved to >0.2 LogMAR in six patients. The complete closure of MH following PPV with inverted ILM technique was eventually achieved in all patients determined by OCT imaging (100%). Only one patient showed recovery of ellipsoid zone and interdigitation zone following the surgery. Elongation of outer nuclear layer was noted in three eyes. The ILM-like membrane not stained with BBG histologically revealed an amorphous structure admixed with GFAP-positive mononuclear cell infiltration. Conclusion: PPV with inverted ILM flap technique achieved 100% closure rates with favorable configuration at an initial surgery in large MHs. Our histopathological data also suggest that even BBG staining-negative membrane may be a useful material for autologous transplantation to the hole.

Purpose: The aims of this study were to analyze optical coherence tomography (OCT) imaging of large macular holes (MHs) treated with inverted internal limiting membrane (ILM) flap technique and to perform a histological examination of an ILM-like membrane tissue obtained during vitrectomy. Patients and methods: This is a retrospective observational case study. Nine patients, comprising of five males and four females, showing large and myopic MHs, underwent pars plana vitrectomy (PPV) with inverted ILM flap technique assisted by brilliant blue G (BBG) staining. Ophthalmological findings including visual acuity and OCT were investigated based on medical records. Formalin-fixed paraffin-embedded tissue section of an ILM-like membrane was submitted for immunohistochemistry with glial fibrillary acidic protein (GFAP). Results: ILM was clearly stained with BBG in eight patients, whereas the ILM in one case revealed no staining with BBG during PPV. Visual acuities improved to >0.2 LogMAR in six patients. The complete closure of MH following PPV with inverted ILM technique was eventually achieved in all patients determined by OCT imaging (100%). Only one patient showed recovery of ellipsoid zone and interdigitation zone following the surgery. Elongation of outer nuclear layer was noted in three eyes. The ILM-like membrane not stained with BBG histologically revealed an amorphous structure admixed with GFAP-positive mononuclear cell infiltration. Conclusion: PPV with inverted ILM flap technique achieved 100% closure rates with favorable configuration at an initial surgery in large MHs. Our histopathological data also suggest that even BBG staining-negative membrane may be a useful material for autologous transplantation to the hole.

Satoru Kase, Wataru Saito, Shohei Mori, Michiyuki Saito, Ryo Ando, Zhenyu Dong, Tomohiro Suzuki, Kousuke Noda, Susumu Ishida Department of Ophthalmology, Hokkaido University Graduate School of Medicine, Sapporo, Japan Purpose: The aims of this study were to analyze optical coherence tomography (OCT) imaging of large macular holes (MHs) treated with inverted internal limiting membrane (ILM) flap technique and to perform a histological examination of an ILM-like membrane tissue obtained during vitrectomy.Patients and methods: This is a retrospective observational case study. Nine patients, comprising of five males and four females, showing large and myopic MHs, underwent pars plana vitrectomy (PPV) with inverted ILM flap technique assisted by brilliant blue G (BBG) staining. Ophthalmological findings including visual acuity and OCT were investigated based on medical records. Formalin-fixed paraffin-embedded tissue section of an ILM-like membrane was submitted for immunohistochemistry with glial fibrillary acidic protein (GFAP).Results: ILM was clearly stained with BBG in eight patients, whereas the ILM in one case revealed no staining with BBG during PPV. Visual acuities improved to >0.2 LogMAR in six patients. The complete closure of MH following PPV with inverted ILM technique was eventually achieved in all patients determined by OCT imaging (100%). Only one patient showed recovery of ellipsoid zone and interdigitation zone following the surgery. Elongation of outer nuclear layer was noted in three eyes. The ILM-like membrane not stained with BBG histologically revealed an amorphous structure admixed with GFAP-positive mononuclear cell infiltration.Conclusion: PPV with inverted ILM flap technique achieved 100% closure rates with favorable configuration at an initial surgery in large MHs. Our histopathological data also suggest that even BBG staining-negative membrane may be a useful material for autologous transplantation to the hole. Keywords: inverted ILM f

Purpose: Echinochrome is a pigment present in the shells and spines of sea urchins. It has been reported to have several biologic protective effects, including in experimental models of myocardial ischemia/reperfusion injury, for which the proposed mechanisms are scavenging reactive oxygen species (ROS) and chelating iron. Endotoxin-induced uveitis (EIU) is an animal model of acute anterior segment intraocular inflammation that is induced by the injection of lipopolysaccharide (LPS). In this study, the therapeutic effect of echinochrome was examined in uveitis using the EIU model. Methods: EIU was induced in Lewis rats via 200 mu g subcutaneous injections of LPS from Escherichia coli. Echinochrome was administered intravenously in 10, 1, or 0.1 mg/kg doses suspended in PBS (controls were injected with PBS only). Twenty-four hours after LPS injection, the number of infiltrating cells and the protein concentration in aqueous humor were determined. Aqueous tumor necrosis factor alpha (TNF-alpha) concentration was quantified with enzyme-linked immunosorbent assay, eyes were stained with nuclear factor (NF) kappa B antibodies, and ROS production was determined by dihydroethidium staining in fresh frozen samples. Results: The number of inflammatory aqueous cells and protein levels were lower in the groups treated with 10 and 1 mg/kg of echinochrome than in the untreated LPS group (p<0.01). Treatment with 10 and 1 mg/kg of echinochrome significantly reduced TNF-alpha concentrations in aqueous humor (p<0.01). The numbers of NF kappa B-positive cells and ROS signals were also reduced by echinochrome administration (p<0.05). Conclusions: Echinochrome ameliorated intraocular inflammation caused by EIU by reducing ROS production, thereby also decreasing the expression of NF kappa B and TNF-alpha. As a natural pigment, echinochrome may therefore be a promising candidate for the safe treatment of intraocular inflammation. The use of sea urchin shells and spines in health

Purpose: Echinochrome is a pigment present in the shells and spines of sea urchins. It has been reported to have several biologic protective effects, including in experimental models of myocardial ischemia/reperfusion injury, for which the proposed mechanisms are scavenging reactive oxygen species (ROS) and chelating iron. Endotoxin-induced uveitis (EIU) is an animal model of acute anterior segment intraocular inflammation that is induced by the injection of lipopolysaccharide (LPS). In this study, the therapeutic effect of echinochrome was examined in uveitis using the EIU model. Methods: EIU was induced in Lewis rats via 200 mu g subcutaneous injections of LPS from Escherichia coli. Echinochrome was administered intravenously in 10, 1, or 0.1 mg/kg doses suspended in PBS (controls were injected with PBS only). Twenty-four hours after LPS injection, the number of infiltrating cells and the protein concentration in aqueous humor were determined. Aqueous tumor necrosis factor alpha (TNF-alpha) concentration was quantified with enzyme-linked immunosorbent assay, eyes were stained with nuclear factor (NF) kappa B antibodies, and ROS production was determined by dihydroethidium staining in fresh frozen samples. Results: The number of inflammatory aqueous cells and protein levels were lower in the groups treated with 10 and 1 mg/kg of echinochrome than in the untreated LPS group (p<0.01). Treatment with 10 and 1 mg/kg of echinochrome significantly reduced TNF-alpha concentrations in aqueous humor (p<0.01). The numbers of NF kappa B-positive cells and ROS signals were also reduced by echinochrome administration (p<0.05). Conclusions: Echinochrome ameliorated intraocular inflammation caused by EIU by reducing ROS production, thereby also decreasing the expression of NF kappa B and TNF-alpha. As a natural pigment, echinochrome may therefore be a promising candidate for the safe treatment of intraocular inflammation. The use of sea urchin shells and spines in health foods an

Purpose: Vascular endothelial growth factor C (VEGF-C) and its receptor VEGFR-3 mediate lymphangiogenesis. In this study, we analyzed the expression of VEGF-C and VEGFR-3 as well as lymphatic vessels in the pterygium and normal conjunctiva of humans. Methods: Fifteen primary nasal pterygia and three normal bulbar conjunctivas, surgically removed, were examined in this study. The lymphatic vessel density (LVD) and blood vessel density (BVD) were determined by the immunolabeling of D2-40 and CD31, markers for lymphatic and blood vessels, respectively. VEGF-C and VEGFR-3 expression in pterygial and conjunctival tissue proteins was detected by Western blotting. Expressions of VEGF-C and VEGFR-3 were evaluated using immunohistochemistry. Results: The LVD was significantly higher in the pterygium than normal conjunctiva (p<0.05). Western blot demonstrated high-level expression of VEGF-C and VEGFR-3 in the pterygium compared with normal conjunctiva. VEGF-C immunoreactivity was detected in the cytoplasm of pterygial and normal conjunctival epithelial cells. The number of VEGF-C-immunopositive cells in pterygial epithelial cells was significantly higher than in normal conjunctival cells (p<0.05). VEGFR-3 immunoreactivity was localized in the D2-40-positive lymphatic endothelial cells. Conclusions: The present findings suggest the potential role of VEGF-C in the pathogenesis and development of a pterygium through lymphangiogenesis and the VEGF-C/VEGFR-3 pathway as a novel therapeutic target for the human pterygium.

Purpose: To investigate the applicability of serine/arginine-rich protein kinase (SRPK)-specific inhibitor, SRPIN340, for attenuation of choroidal neovascularization (CNV) formation using a mouse model. Methods: Laser photocoagulation was performed to induce CNV in C57BL/6J mice, followed by intravitreal injection of SRPIN340 or vehicle. Seven days after the treatment, the CNV size was evaluated using a flatmount technique. Protein levels of vascular endothelial growth factor (VEGF) and inflammation-associated molecules, such as monocyte chemoattractant protein (MCP)-1 and intercellular adhesion molecule (ICAM)-1, in the retinal pigment epithelium-choroid complex were measured with enzyme-linked immunosorbent assay. Expression levels of total Vegf, exon 8a-containing Vegf isoforms, and F4/80 (a specific marker for macrophage) were assessed using real-time PCR. Results: SRPIN340 inhibited CNV formation in a dose-dependent manner. Compared with the vehicle, SRPIN340 significantly decreased the protein levels of VEGF, MCP-1, ICAM-1, and consequently inhibited macrophage infiltration. Furthermore, SRPIN340 suppressed the gene expression levels of total Vegf and exon 8a-containing Vegf isoforms. Conclusions: SRPIN340, a specific inhibitor of SRPK, suppressed Vegf expression and attenuated CNV formation. Our data suggest the possibility that SRPIN340 is applicable for neovascular age-related macular degeneration as a novel chemical therapeutics.

Purpose: Vascular endothelial growth factor C (VEGF-C) and its receptor VEGFR-3 mediate lymphangiogenesis. In this study, we analyzed the expression of VEGF-C and VEGFR-3 as well as lymphatic vessels in the pterygium and normal conjunctiva of humans. Methods: Fifteen primary nasal pterygia and three normal bulbar conjunctivas, surgically removed, were examined in this study. The lymphatic vessel density (LVD) and blood vessel density (BVD) were determined by the immunolabeling of D2-40 and CD31, markers for lymphatic and blood vessels, respectively. VEGF-C and VEGFR-3 expression in pterygial and conjunctival tissue proteins was detected by Western blotting. Expressions of VEGF-C and VEGFR-3 were evaluated using immunohistochemistry. Results: The LVD was significantly higher in the pterygium than normal conjunctiva (p<0.05). Western blot demonstrated high-level expression of VEGF-C and VEGFR-3 in the pterygium compared with normal conjunctiva. VEGF-C immunoreactivity was detected in the cytoplasm of pterygial and normal conjunctival epithelial cells. The number of VEGF-C-immunopositive cells in pterygial epithelial cells was significantly higher than in normal conjunctival cells (p<0.05). VEGFR-3 immunoreactivity was localized in the D2-40-positive lymphatic endothelial cells. Conclusions: The present findings suggest the potential role of VEGF-C in the pathogenesis and development of a pterygium through lymphangiogenesis and the VEGF-C/VEGFR-3 pathway as a novel therapeutic target for the human pterygium.

Purpose: To investigate the applicability of serine/arginine-rich protein kinase (SRPK)-specific inhibitor, SRPIN340, for attenuation of choroidal neovascularization (CNV) formation using a mouse model. Methods: Laser photocoagulation was performed to induce CNV in C57BL/6J mice, followed by intravitreal injection of SRPIN340 or vehicle. Seven days after the treatment, the CNV size was evaluated using a flatmount technique. Protein levels of vascular endothelial growth factor (VEGF) and inflammation-associated molecules, such as monocyte chemoattractant protein (MCP)-1 and intercellular adhesion molecule (ICAM)-1, in the retinal pigment epithelium-choroid complex were measured with enzyme-linked immunosorbent assay. Expression levels of total Vegf, exon 8a-containing Vegf isoforms, and F4/80 (a specific marker for macrophage) were assessed using real-time PCR. Results: SRPIN340 inhibited CNV formation in a dose-dependent manner. Compared with the vehicle, SRPIN340 significantly decreased the protein levels of VEGF, MCP-1, ICAM-1, and consequently inhibited macrophage infiltration. Furthermore, SRPIN340 suppressed the gene expression levels of total Vegf and exon 8a-containing Vegf isoforms. Conclusions: SRPIN340, a specific inhibitor of SRPK, suppressed Vegf expression and attenuated CNV formation. Our data suggest the possibility that SRPIN340 is applicable for neovascular age-related macular degeneration as a novel chemical therapeutics.